Electron-event representation data enable efficient cryoEM file storage with full preservation of spatial and temporal resolution

Abstract

Direct detector device (DDD) cameras have revolutionized electron cryomicroscopy (cryoEM) with their high detective quantum efficiency (DQE) and output of movie data. A high ratio of camera frame rate (frames per second) to camera exposure rate (electrons per pixel per second) allows electron counting, which further improves the DQE and enables the recording of super-resolution information. Movie output also allows the correction of specimen movement and compensation for radiation damage. However, these movies come at the cost of producing large volumes of data. It is common practice to sum groups of successive camera frames to reduce the final frame rate, and therefore the file size, to one suitable for storage and image processing. This reduction in the temporal resolution of the camera requires decisions to be made during data acquisition that may result in the loss of information that could have been advantageous during image analysis. Here, experimental analysis of a new electron-event representation (EER) data format for electron-counting DDD movies is presented, which is enabled by new hardware developed by Thermo Fisher Scientific for their Falcon DDD cameras. This format enables the recording of DDD movies at the raw camera frame rate without sacrificing either spatial or temporal resolution. Experimental data demonstrate that the method retains super-resolution information and allows the correction of specimen movement at the physical frame rate of the camera while maintaining manageable file sizes. The EER format will enable the development of new methods that can utilize the full spatial and temporal resolution of DDD cameras.

Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs

“…The slit width of the energy filter was set to 10eV. 5599 movie stacks containing 1429 raw frames were collected with EER (electron event representation) mode 48 of Falcon 4 detector at a magnification of 165 kX corresponding to a pixel size of 0.727 Å. Each movie stack was recorded with an exposure time of 6s with a total dose of 50e/A2 on sample and a defocus range between 0.5 to 1.2 µm.…”

A data reduction and compression description for high throughput time-resolved electron microscopy

“…A recent study 8 has proposed storing L4 reduced data in a sparse format to benefit from higher dose fractionation without overwhelming acquisition systems with storage requirements. To achieve super-resolution electron counting, which is critical for improving reconstruction resolution in cryo-EM, they propose subdividing each pixel before counting and storing the higher-resolution spatial locations of electron events using a higher bit-depth.…”

“…In single-particle analysis (SPA) the energy deposited by inelastically scattered electrons manifests as sample damage and ice drift, where global and site-specific sample damage is detectable even at exposures as low as 0.1 e/ Å 2 26 . Here higher electron dose fractionation improves resolution in two ways: (1) by reducing coincidence loss and thereby improving detection efficiency 27 and (2) by enabling more accurate estimation of sample drift at a higher temporal resolution 8 . Increasing detector frame rates can reduce the average displacement of each particle captured in each dose-fractionated frame, but doing so further inflates the already large amounts of movie-mode data collected (see Supplementary Fig.…”

“…Here, the imaging resolution is demonstrably improved when fast detectors fractionate the total electron dose on a sample onto a time series of micrographs that are individually corrected for relative dose-induced motion 7 . In fact, recent work suggests that using more efficient data representation to further increase dose fractionation, hence finer time resolution, can improve spatial resolution 8 .…”

Setting up and operating a cryo-EM laboratory

“…For example, four high-end electron microscopes equipped with electron-counting cameras would need a storage system with at least a petabyte (PB) capacity. Larger and faster electron counting cameras are now coming onto the market, but at the same time, data formats are being developed such as electron event recording (Guo et al ., 2020) that allow highly efficient storage of image information. Nevertheless, we can expect further significant growth in the requirements for data storage of cryo-EM data.…”